Project description:Andrographis paniculata Nees and its major compound andrographolide is known to exhibit a pleothera of activities. Active component enriched extracts are known to provide various beneficial effects, and therefore it was interesting to investigate whether increased amount of major compound alters gene expression. To acheive this, HePG2 cells treated with Andrographis paniculata extract (AP) and andrographolide enriched –extract (AP20) were subjected to microarray analysis to check their effect on global gene expression.
Project description:Andrographis paniculata (AP) is a medicinal herb used to treat infectious diseases. The medicinal properties are due to presence of andrographolide and other secondary metabolites synthesized via mevalonic acid (MVA) and methyl erythritol phosphate (MEP) pathways. To increase the andrographolide content plants were treated with 50µM Jasmonic acid (JA) for one week. HPLC analysis revealed that one fold increase in the andrographolide content compared to control. To study the signal mechanism underlying in the stress response, samples were subjected to Q-TOF-LC-MS/MS analysis. JA influences the expression of MVA and MEP enzymes in treated (22 enzymes) than in untreated (3 enzymes). We found total 3308 proteins in control and 3994 in elicited sample in which 1393 were commonly found in both. The differential expression revealed 621 proteins were down-regulated and 201 proteins were up regulated. The functional annotation involved 40 metabolic processes where post translational modifications was highly up regulated in treated (5.7%). The total proteome of A.paniculata was reported for the first time. The comparison of elicited and non elicited (control) samples revealed that the elicitation is an integrated process which release variety of bioactive compounds to defend itself from variety of pathogens. The Knowledge obtained from this scrutiny, JA dependent stress adaptive response is likely to have industrial and agricultural impact. It creates a hope towards future for the development of eco-friendly ways of managing pests and tapping into a largely unexplored treasure of plant-derived bioactive metabolites for human use. Particularly in this plant, it is useful for the production of medicines to cure different kinds of diseases and disorders.
Project description:To shed light on the biosynthesis mechanisms of these diterpenes, we performed the first high-throughput RNA-seq for A. paniculata leaves with time-serialized MeJA treatments. The transcriptomic approach not only defined a comprehensive transcriptome (comprised of 20,629 highly expressed genes), but also profiled time-serialized gene expression patterns with 4,463 up-regulated genes and 6,517 down-regulated genes responding to MeJA induction. These up-regulated genes significantly enriched in biological processes like stress responses, metabolic pathways, and secondary metabolites biosynthesis, especially terpene/terpenoid metabolic process. Consistent with the gradual accumulation of andrographolide, the expression of most genes involved in MEP pathway showed progressive increase. In the hypothetical terpenoid metabolism pathway, ApCPS2, which was putatively involved in andrographolide synthesis, dramatically increased its expression. Among other co-regulated genes, 154 genes encode cytochrome P450s (CYP450s); 42 genes were predicted as UDP glycosyltransferases (UGTs); and 363 genes belong to transcription factors.
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.